CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Summary

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

Abstract

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

Introduction

The majority of biological processes happening within a cell are thought to be carried out by protein assemblies rather than single proteins¹. Consequently, in order to elucidate the specific biological role of a protein subunit in a cell it is necessary to understand its structural interactions with other proteins or ligands in complexes². However, studying proteins natively, maintaining their non-covalent interactions and activity, remains challenging. One of the shortcomings of native protein studies is a suitable native separation technique that is compatible with various downstream protein analysis techniques. Therefore, recent interest in s....

Protocol

Note: This video protocol is based on an associated publication⁹. Specific reagents, material and equipment necessary to perform all the steps described in this protocol are listed in the materials section. The recipes and information for preparation of all required solutions are itemized in Table 1.

1. Pouring of Gradient Tube Gels

1. Assembling the Casting System
   1. Using a flame-heated razor blade or scalpel, cut a 20 ml serological pipette along an orthogonal section 10 cm from the top of the dispensing tip. Ensure that the cut surface is smooth. Keep the bottom piece (with the tip) and disc....

Results

200 µg of proteins extracted from cryogenically ground mouse hearts were fractionated natively into 14 fractions of 150 µl each, using the CN-GELFrEE method in a 1-12% T gel tube. An aliquot of 10 µl of each fraction was run on a CN-PAGE slab gel and silver stained. A clear depiction of the fractionation and resolution obtained with CN-GELFrEE fractionation is shown in Figure 2A. Fractions were collected over two hours and the gel assay shows a consistent pattern of incre.......

Discussion

CN-GELFrEE is derived from the clear native (CN) PAGE buffer system that uses a mixture of anionic and neutral detergents as carrier molecules⁹ for molecular-weight-based protein fractionation through a gel matrix. The application of CN-GELFrEE to a native mouse heart protein extract generated low complexity fractions with discrete bandwidths while maintaining non-covalent interactions of biomolecular assemblies. Comparison of fractions between CN-PAGE and reducing SDS-PAGE slab gels showed mass shifts that de.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
30% acrylamide/bis solution, 37.5:1	Bio-Rad	161-0158	This solution is neurotoxic.
6-aminohexanoic acid (ε-aminocaproic acid)	Sigma-Aldrich	A2504	This chemical is an irritant.
Ammonium persulfate (APS)	Fisher Scientific	7727-54-0	This chemical is flammable, toxic, and corrosive.
Coomassie blue G250	Bio-Rad	161-0406
Dodecylmaltoside	Sigma-Aldrich	D4641	This chemical is an irritant.
Ethylene glycol tetraacetic acid (EGTA)	FLUKA	3777	This chemical is toxic.
Ethylenediamine tetraacetic acid (EDTA)	Fisher Scientific	BP120	This chemical is toxic.
Glycerol	Fisher Scientific	56-81-5
Imidazole	Sigma-Aldrich	I2399
Liquid nitrogen	Any supplier	n/a	Store liquid nitrogen in containers designed for low-temperature liquids
Mouse heart	Bioreclamation LLC	MSE-HEART
n-butanol	Fisher Scientific	71-36-3	This chemical is flammable and toxic.
Ponceau S	Sigma-Aldrich	BP103
Protease Inhibitor Cocktail	Thermo Fisher Scientific	78430	This chemical is toxic.
Sodium deoxycholate	Sigma-Aldrich	30970	This chemical is an irritant.
Sucrose	JTBaker	4097
Tetramethylethylenediamine (TEMED)	Bio-Rad	161-0800	This chemical flammable and irritant.
Tris-HCl	Amresco	M151
Trycine	Bio-Rad	161-071
Equipment
Gradient mixer	Hoefer	SG15
Magnetic stir	Any supplier	n/a
Magnetic bars	Any supplier	n/a	Small enough to fit inside the gradiet mixer chambers.
Power supply	Bio-Rad	164-5056	Voltage up to 1,500 V
Refrigerated centrifugue	Eppendorf	022622552	Up to 15,000 x g
Materials
15 ml conical tube	Any supplier	n/a
30 kDa-MWCO ultra centrifugal filter	Millipore	UFC503096
Flexible tubing	Saint-Gobain	B000FOV1J0	Approximately 1 m length
Ice bucket	Any supplier	n/a
Liquid/air metallic or plastic valve. (e.g. aquarium air/water flow control lever valve)	Any supplier	n/a	Aquarium valves are compatible.
Mortar and pestle	Any supplier	n/a
Native PAGE 3-12% T slab gel	Invitrogen	BN1003
Parafilm	Bemis	PM-996
Petri dish	Fisher Scientific	08-757-13
Protein low bind tube 1.5 ml	Eppendorf	022431081
Razor blade	IDL Tools	TE05-091C
Regenerated cellulose dialysis tubing 3,500 MWCO	Fisher Scientific	21-152-9
SDS-PAGE slab gel for any kDa	Bio-Rad	456-9036
Serological pipets (25 ml)	VWR	89130-900
Syringe (10 ml)	Any supplier	n/a